Abstract number: 636. 24hrs. 12hrs. A. B.
Novel antitumor quinols: the role of thioredoxinEng-Hui Chew, Jenny C. Cookson, Charles S. Matthews, Ji-Hong Zhang, Thilo Hagen, Tracey D. Bradshaw, Malcolm F.G. Stevens, Andrew D. WestwellCentre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK
Novel antitumor QuinolsAW464 and BW114 exhibit selective antiproliferative activity against colon, renal and breast carcinoma cell lines (GI50 < 0.5 μM). Growth inhibition against renal, colon and breast xenografts has also been demonstrated1, 2. Preliminary results (microarray analysis, insulin reduction assay) suggested that quinols targeta 12 kDa redox-regulating thiol protein thioredoxin (Trx)3. Here we report further evidence of quinols interacting with thioredoxin and the results of investigations into the induction of oxidative stress and apoptosis by quinols.
Figure 4: Annexin V-FITC/Propidium Iodide double staining
Figure 1: A, B: Mass spectrum of human Trx
Figure 3: A, B: MTT cell viability
Figure 2: ROS production induced by AW464
HCT116 colon cells treated with AW464 (1 µM) showed an increasing proportion of cells undergoing apoptosis between 12 and 24 hours.
Figure 5: Western blot
Dose-dependent PARP cleavage and caspase 3 activation was observed in HCT116 cells treated with AW464 and BW114 for 24 hours.
Campothecin 1 µM, 24hrs (tve ctrl)
1 3 6 0.5 1 3
Reduced Trx + AW464
ROS production was induced in HCT116 colon cells following 7 hours AW464 treatment.
Cleaved Caspase 3
Modulation of the glutathione (GSH) system and quinol cytotoxicity.
- BSO-induced GSH depletion increased cytotoxicity of quinols by 10-fold (Fig3A).
- Supplementation of GSH monoethyl ester
caused a 2-fold reduction in potency (Fig3B).
No blockade of cytotoxicity by pan caspase inhibitor Z-VAD-FMK and caspase 3 inhibitor Z-DEVD-FMK, suggesting that quinols induce apoptosis via both caspase dependent and independent pathways.
(A) Non-drug treated thioredoxin: mass peak 11,606.
(B) AW464 treated thioredoxin: mass peaks 12,336, 12,586
and 12,821 corresponding to thioredoxin plus 3, 4 and 5
AW464 molecules respectively.
Subsequent MS/MS analysis of trypsin digested peptides
showed additional mass of AW464 only at cysteine residues.
We conclude that quinols induce oxidative stress, cellular apoptosis and thioredoxin emerges as a molecular target of these novel antitumor agents.
1. Wells et al J. Med. Chem. 2003, 46, 532.
2. Berry et al J. Med.Chem. 2005, 48, 639.
3. Bradshaw et al Cancer Res. 2005, 65, 3911.